Abstract

Alternative splicing of the pyruvate kinase M gene involves a choice between mutually exclusive exons 9 and 10. Use of exon 10 to generate the M2 isoform is crucial for aerobic glycolysis (the Warburg effect) and tumour growth. We previously demonstrated that splicing enhancer elements that activate exon 10 are mainly found in exon 10 itself, and deleting or mutating these elements increases the inclusion of exon 9 in cancer cells. To systematically search for new enhancer elements in exon 10 and develop an effective pharmacological method to force a switch from PK-M2 to PK-M1, we carried out an antisense oligonucleotide (ASO) screen. We found potent ASOs that target a novel enhancer in exon 10 and strongly switch the splicing of endogenous PK-M transcripts to include exon 9. We further show that the ASO-mediated switch in alternative splicing leads to apoptosis in glioblastoma cell lines, and this is caused by the downregulation of PK-M2, and not by the upregulation of PK-M1. These data highlight the potential of ASO-mediated inhibition of PK-M2 splicing as therapy for cancer.

Antisense oligonucleotide (ASO) walk along the 10W region. (a) Diagram of the PK-M genomic region, and the RT-PCR assay to measure M1/M2 ratios. This region comprises introns 8, 9 and 10 (represented by the lines), intact exon 9 (green box), exon 10 (red box) and portions of exons 8 (white box) and 11 (black box). Numbers above the boxes show the length in nucleotides. Primers annealing to exons 8 and 11 used to amplify the endogenous PK-M transcript are represented by the arrows. cDNA amplicons generated after radioactive PCR are shown below and labelled accordingly. Three spliced species were observed: the shorter double-skipped species, comprising only exons 8 and 11 (D, 271 nt); M1, including exon 9 (A, 398 nt); and M2, including exon 10 (B, 398 nt). To distinguish between M1 and M2, a subsequent PstI digest was carried out. Only M2 has a PstI site, resulting in two cleavage products: B1 (213 nt) and B2 (185 nt), which are the 3′ and 5′ ends of M2, respectively. (b) Scheme of the ASO screen focused on the 10W region. The sequence of exon 10 from 25 to 88 nt upstream of the 5′ splice site (ss) is indicated in red. Stacked lines represent individual ASOs and are aligned to the complementary sequence in exon 10. The ASO names are indicated on the left, with the subscript numbers indicating the target-sequence coordinates. The initial ASO walk is represented at the top, with ASO 10W45–59 indicated in red and shown to be annealing to the 10W region (bounded by the rectangle) by vertical dashed lines. The microwalk ASOs are indicated below, and the complementary sequence targeted by ASO 10M46–60 is indicated with vertical dashed lines. (c,d) ASO walks. Radioactive RT-PCR and restriction digest of endogenous PK-M transcripts in HEK-293 cells after transfection of ASOs. Initial walk ASOs, transfected at 30 nM, are shown in (c), and microwalk ASOs transfected at 60 nm are shown in (d). RNAs were harvested from cells 48 h after transfection. The transfected ASO is indicated at the top. cDNA amplicons and fragments are indicated on the left. Lane numbers and quantifications are indicated at the bottom. Each product was quantified as a percentage of the total of M1, M2 and double-skipped species. %M1 and %M2 are shown. All standard deviations are ≤4% (n = 3).

Characterizing the 10W ASO target region. (a) Scheme of method used to duplicate the exon 10 10W region into exon 9 in a minigene. The minigene comprises the same genomic region as indicated in a. To amplify minigene transcripts, we used a primer annealing to a vector-specific sequence upstream of the genomic insert, pcDNAF []. Minigene mutant names are indicated below. The indicated exon 9 (green) nucleotides at the top were mutated to the corresponding exon 10 (red) sequences on the right. The 10W minigene duplicates the entire exon 10 10W region into exon 9; the 10F minigene duplicates the first 8 nt of 10W45–59; and the 10B minigene duplicates the last 7 nt of 10W45–59. The ASOs that target 10W and the flanking regions are indicated below. (b) The 10W region is an exon 10 ESE. Mutant minigenes were analysed by transient transfection into HEK-293 cells, followed by radioactive RT-PCR and restriction digestion, as in . Constructs from (a) are labelled at the top. Labelled bands are indicated in lower case on the left and right, with important bands in blue font. %M1 is indicated at the bottom. Bands are as follows: uncut M1 fragment (a, 481 nt); uncut M2 fragment (b, 481 nt); PstI-cleaved M2 5′ fragment (b2, 268 nt); PstI-cleaved M2 3′ fragment (b3, 213 nt); a spliced mRNA that skips both exons 9 and 10 (d, 314 nt); an exon 9–exon 10 doubly included mRNA expressed from the 10B minigene (lanes 5 and 6) is indicated on the left (f, 648 nt). This band is sensitive to PstI (f1, 435 nt). Standard deviations (s.d.) are 0.2%, 0.3% and 2.6% for 10G, 10F and 10B, respectively; n = 3. (c,d) Minigene transcript-level changes as a result of ASO co-transfection in HEK-293 cells. ASOs were transfected at a nominal final concentration of 60 nM. The wild-type (c) and exon 10 duplication (d) minigenes [], together with the identity of the ASOs, are indicated at the top. Labelled bands are indicated in lower case on the left, with important bands in blue font. The exon 10–exon 10 doubly included mRNA in (d) expressed from the exon 10 duplication minigene is indicated on the right (g, 648 nt). %M1, %M2 or %Skp is indicated at the bottom. Standard deviations for (c) are 0.6%, 4.2% and 2.9% for control, 10W45–59 and 10M46–60, respectively; s.d. for (d) are 0.8%, 9.4% and 2.6% for control, 10W45–59 and 10M46–60, respectively; n = 3. (e) Diagram of a homologous potential cross-hybridizing region for ASOs 10W45–59 and 10M46–60 ASOs. Alignment of the sequences of ASO 10W45–59, the complementary region in exon 10 (indicated in red), and a homologous region in intron 9 (indicated in blue, 129–156 nt upstream of the exon 9 5′ss) is shown. Vertical lines indicate sequence identity. Diagram of minigene mutants used in (f–h) is shown on the left. d10W has the 10W ASO binding site in exon 10 removed and replaced by the corresponding region in exon 9. dInt9 has a 15 nt deletion of the homologous intron 9 region (e). dM has both mutations. (f–h) Minigene transcript-level changes as a result of ASO co-transfection in HEK-293 cells. ASOs were transfected at a final nominal concentration of 60 nM. The minigenes, together with the identity of ASOs, are indicated at the top. Labelled bands are indicated in lower case on the left, with important bands in blue font. ASOs and minigenes from (e) are indicated at the top, %M1 is indicated at the bottom and bands are indicated on the left. %M1, %M2 or %Skp is indicated at the bottom. Standard deviations for (f) are 1.2%, 1.6% and 1.5%; for (g) they are 0.4%, 2.0%, 3.9%; and for (h) they are 0.2%, 0.6% and 0.6%, corresponding to control, 10W45–59 and 10M46–60 ASOs, respectively; n = 3.

Effects of ASOs on PK-M mRNA and protein levels in glioblastoma cells. (a,c) Effects of ASOs 10M46–60, 10W45–59 and 10MS139–153 on endogenous PK-M mRNAs in (a) A172 and (c) U87-MG glioblastoma cells. Radioactive RT-PCR and restriction digest of endogenous PK-M transcripts were performed for the indicated cell lines 36 h after transfection of 30, 60 or 90 nM 10W45–59 or 10M46–60 ASO, or 60 or 90 nM 10MS139–153 ASO. The control ASO was transfected at 90 nM. %M1, %M2 and %Skp are indicated at the bottom. All s.d. are ≤4%; n = 3. (b,d) Immunoblot analysis of PK-M protein isoform levels in (b) A172 and (d) U87-MG cells transfected in (a,c). A representative blot from one of three independent experiments is shown. Antibodies used are indicated on the left.

Effects of ASOs on glioblastoma cells. (a,b) Exon 10 ASOs induce apoptosis in glioblastoma cells. (a) U87-MG or A172 cells were transfected with the indicated ASOs at a nominal final concentration of 90 nM, stained with Annexin V-APC/7-AAD 36 h after transfection and analysed by flow cytometry. The percentage of Annexin-V-positive cells is indicated for the two right quadrants in each plot. Each plot is a representative of three biological replicates. (b) ASO-induced apoptosis is dose-dependent. The indicated cells were transfected with ASOs 10W45–59 or 10M46–60 at 30, 60, or 90 nM, or ASO 10MS139–153 at 60 or 90 nM. The control (Ctl) ASO was transfected at 90 nM. The percentage of Annexin-V-positive cells is plotted for each condition. The identity and dose of ASOs are indicated below the x-axis. Error bars represent s.d. (n = 3). (c,d) Role of PK-M1 protein isoform in apoptosis induction. (c) Immunoblot analysis of A172 cells stably transduced with rtTA and doxycycline (dox)-inducible human T7-tagged PK-M1 cDNA. Cells were grown in parallel, with or without doxycycline, and harvested after 72 h. Antibodies used are indicated on the left. (d) Cells were grown as in (c) and then transfected with the indicated ASOs at 60 nM. Cells were then stained and analysed for Annexin V 36 h after transfection. Histograms indicate the percentage of Annexin-V-positive cells for each condition. Doxcycyline on/off conditions are indicated on the left. Error bars represent s.d. (n = 3). (e,f) Role of PK-M2 protein isoform in apoptosis induction. A172 or U87 cells were stably transduced with T7-tagged human PK-M2 cDNA; transductants and the parental cell lines were transfected with the indicated ASOs at a nominal final concentration of 90 nM. (e) Immunoblot analysis of cells transfected with the indicated ASOs. Antibodies used are indicated on the left. (f) ASO-transfected cells were analysed for Annexin V as in (d). Histograms indicate the fold increase in Annexin-V-positive cells, compared with control ASO, for each cell line. Error bars represent s.d. (n = 3).